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Isolation, incubation, and parallel functional testing and identification by
FISH of rare microbial single-copy cells from multi-species mixtures using the
combination of chemistrode and stochastic confinement†
Weishan Liu, Hyun Jung Kim, Elena M. Lucchetta, Wenbin Du and Rustem F. Ismagilov*
Received 11th March 2009, Accepted 1st May 2009
First published as an Advance Article on the web 14th May 2009
DOI: 10.1039/b904958d
This paper illustrates a plug-based microfluidic approach combining the technique of the chemistrode
and the principle of stochastic confinement, which can be used to i) starting from a mixture of cells,
stochastically isolate single cells into plugs, ii) incubate the plugs to grow clones of the individual cells
without competition among different clones, iii) split the plugs into arrays of identical daughter plugs,
where each plug contained clones of the original cell, and iv) analyze each array by an independent
technique, including cellulase assays, cultivation, cryo-preservation, Gram staining, and Fluorescence
In Situ Hybridization (FISH). Functionally, this approach is equivalent to simultaneously assaying the
clonal daughter cells by multiple killing and non-killing methods. A new protocol for single-cell FISH,
a killing method, was developed to identify isolated cells of Paenibacillus curdlanolyticus in one array of
daughter plugs using a 16S rRNA probe, Pc196. At the same time, live copies of P. curdlanolyticus in
another array were obtained for cultivation. Among technical advances, this paper reports
a chemistrode that enables sampling of nanoliter volumes directly from environmental specimens, such
as soil slurries. In addition, a method for analyzing plugs is described: an array of droplets is deposited
on the surface, and individual plugs are injected into the droplets of the surface array to induce
a reaction and enable microscopy without distortions associated with curvature of plugs. The overall
approach is attractive for identifying rare, slow growing microorganisms and would complement
current methods to cultivate unculturable microbes from environmental samples.
Introduction
This paper describes using a combination of chemistrode and
stochastic confinement to isolate individual microbial cells from
diverse mixtures into plugs, then incubate these isolated cells to
provide growth without competition, and finally split the
resulting plugs containing cells into multiple daughter plugs,
each containing clones of the original cell. These daughter plugs
can then be used for multiple analyses, such as identification and
functional testing, in parallel.
Isolation and functional characterization of microbes from
diverse multi-species mixtures is of wide interest because these
mixtures perform critical functions in environments ranging
from soils, to oceans, to niches inside a eukaryotic host. Metagenomic efforts are documenting the genetic diversity of these
mixtures,1–6 but these methods do not provide access to live cells.
Furthermore, short reads do not provide information on which
microorganism has which genes.7 Methods for identifying cells of
a specific species within a mixture, for example Fluorescence In
Situ Hybridization (FISH), may be used to identify bacteria
having particular genes,8,9 but FISH is a killing method and does
not provide live, isolated microorganisms.
Department of Chemistry and Institute for Biophysical Dynamics, The
University of Chicago, 929 East 57th Street, Chicago, Illinois, 60637,
USA. E-mail: [email protected]; Fax: +1 773-834-3544; Tel:
+1 773-702-5816
† Electronic supplementary information (ESI) available: Supplementary
methods, Fig. S1–S4 and Table S1. See DOI: 10.1039/b904958d
This journal is ª The Royal Society of Chemistry 2009
Isolation of microorganisms is difficult because many of them
appear to be unculturable in traditional experiments, although
new techniques are rapidly improving cultivability.10–12 One
problem with culturing rare cells is that they may grow slowly,
and, when grown in a mixture, they get out-competed by other
species that grow more rapidly or are present at a higher density
initially.13 Limiting dilution can be used to address this problem:
by diluting the original mixture to such an extent that individual
aliquots (e.g. 50 mL aliquots placed into wells of a well plate)
contain individual cells, competition is eliminated.14,15 However,
dilution to such a low density makes it more difficult to detect
organisms that grow very slowly, undergo just a few divisions, or
grow to a low final density.15,16 In addition, analysis of secreted
molecules, such as cellulase enzymes needed for biomass
conversion in production of biofuels, becomes more difficult
when starting with a low density of cells, because secreted
molecules are initially present at low concentrations. Finally,
such dilution greatly changes the original microenvironment of
the sample and may negatively affect the growth of the organism.
Gel microdroplets (GMDs) have been used successfully to
overcome some of these problems and confine individual cells of
microorganisms in small volumes and enhance growth.10,17
The microfluidic technique described here builds on these
previous advances. Here we use the chemistrode18,19 and
stochastic confinement in arrays of nanoliter plugs12,20–24 to
construct a system suitable for isolating rare cells. Plugs are
aqueous droplets surrounded by an immiscible fluorocarbon
carrier fluid,25,26 and they are useful for monitoring growth of
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cells and for analyzing their secretions.22,23,27,28 The chemistrode
relies on multiphase aqueous/fluorous flow to deliver stimuli or
to sample responses by trapping a solution into plugs.18 Separation of competing species into plugs eliminates competition for
nutrients and allows all species to grow. Isolation may also
prevent toxic compounds released by one species from contacting
the other species. Here, isolating cells in plugs provides several
other attractive features: i) it eliminates scattering from gels,
simplifying analysis by microscopy; ii) it provides an opportunity
to confine and analyze secreted molecules,20,22,27,28 from the level
of a few cells all the way down to the single-cell level;29 and iii)
after a few divisions of the original cell within a plug, it enables
splitting18,30 of this plug into daughter plugs, each containing
clones of the original cell. This latter feature allows each of the
daughter plugs to be manipulated or analyzed by an independent
technique. For example, small clonal populations can be
analyzed by mutually incompatible techniques, such as those that
kill cells vs. those requiring live cells.
In this paper, we present a version of the chemistrode that
enables sampling directly from the environment, even from
heterogeneous samples, such as a soil slurry. We then illustrate
that the chemistrode can be combined with stochastic confinement to i) stochastically isolate rare cells in a mixture with more
abundant species into plugs, ii) incubate the plugs to grow
colonies of the isolated cells, iii) split the plugs into arrays of
identical daughter plugs, and iv) perform multiple tests in
parallel. We demonstrate this approach by using a mixture of
Paenibacillus curdlanolyticus, a cellulase-producing species, as
the rare and slow-growing species, and E. coli as the abundant
and rapid-growing species. We also develop a protocol to identify P. curdlanolyticus in plugs by using FISH.
space between the silica capillary and the Teflon tubing. A
negative pressure was applied on the 100 mm I. D. Teflon tubing
by aspirating at 0.9 mL min1. Both the carrier fluid and the
aqueous sample were aspirated into this Teflon tubing, and the
aqueous sample was segmented into plugs by the carrier fluid at
the entrance.
Cultivation of microorganisms and culture media
Bacterial species of Paenibacillus curdlanolyticus (ATCC 51899)
and Escherichia coli (E. coli, ATCC 25922) were obtained from
the American Type Culture Collection. P. curdlanolyticus cells
were enriched in 30 g L1 of sterilized trypticase soy broth (TSB)
media (BD Company) at 30 C for 12 h, and E. coli cells were
enriched in Difco Luria-Bertani (LB) broth media (BD
Company) at 37 C for 3 h. Seed inoculum of each species was
cultured in a rotary shaking incubator (SI-600 Lab Companion,
Jeio Tech) at 180 rpm. During the seed culture of P. curdlanolyticus, cellulases were induced by adding filter-sterilized (0.45
mm, Whatman) carboxymethyl-cellulose (CM-cellulose; sodium
salt, 0.7 D.S., Sigma-Aldrich) at 1 g L1 final concentration.
The green fluorescence protein (GFP)-labeled E. coli (a mutant
E. coli strain containing PUCP24/EGFP plasmids in E. coli K12
YMel-1 host) was constructed in the laboratory. The red fluorescence protein (RFP)-labeled E. coli (a mutant E. coli strain
containing DsRed encoding plasmids in E. coli DH10B host) was
provided by Professor Benjamin Glick of the University of
Chicago. GFP-labeled E. coli was cultured in Difco tryptic soy
agar (TSA) media (BD company) that included 100 mg L1
kanamycin and 20 mg L1 gentamicin. RFP-labeled E. coli was
cultured in TSA media that included 100 mg L1 ampicillin.
Preparation of live cells
Experimental
Design and manipulation of the chemistrode for environmental
sampling
A tip of a piece of glass septum theta tubing (1.5 mm, World
Precision Instruments) was made sharp by fusing, and then
inserted into a piece of Tygon tubing (250 mm I. D., 2000 mm O.
D.) (Fig. S1†). Then a piece of a standard, polyimide coated,
flexible, fused silica capillary (318 mm I. D., 435 mm O. D.,
Polymicro) was inserted into the other end of the piece of
Tygon tubing. A piece of Teflon tubing (100 mm I. D., 150 mm
O. D., Zeus) was inserted into the open end of the glass septum
theta tubing, through the Tygon tubing, all the way down to the
distal tip of the silica capillary. A second piece of Teflon tubing
(200 mm I. D., 250 mm O. D., Zeus) was also inserted into the
open end of the glass tubing, and this tubing extended only
down to the proximal tip of the glass tubing. The gap between
the two pieces of Teflon tubing and the glass tubing was filled
with half-cured polydimethylsiloxane (PDMS) glue (DowCorning Sylgard 184 A and B at a ratio of 10 : 1, cured at 110
C for 110 s), and then the device was baked at 65 C for the
PDMS glue to fully cure.
For sampling, the chemistrode was held by the Tygon tubing,
and the tip was dipped into the aqueous sample. The carrier fluid
was delivered from the 200 mm I. D. Teflon tubing at 0.5 mL
min1, and it flowed to the tip of the chemistrode through the
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An inoculum of either P. curdlanolyticus or E. coli was cultured in
either TSB (for P. curdlanolyticus) or LB (for E. coli) media
respectively. Seed inoculum of either GFP-labeled E. coli or RFPlabeled E. coli was cultured in either LB media including 100 mg
L1 kanamycin (for GFP-labeled E. coli) or LB media including
100 mg L1 ampicillin (for RFP-labeled E. coli) respectively. Cells
in a seed culture were harvested at the exponential phase, and then
the cells were washed twice with autoclaved 0.9% (w/v) NaCl
solution. For immediate use, the number of live cells for each
species was approximately estimated by either counting cells
under an epi-fluorescence microscope (DMI 6000 B, Leica) (for
GFP-labeled E. coli and RFP-labeled E. coli), or by staining with
live/dead fluorescent dye (Live/Dead BacLight Bacterial viability
kit, Molecular Probes) then counting cells (for P. curdlanolyticus
and E. coli), or by measuring optical density through a UV/Vis
spectrophotometer (Agilent) for P. curdlanolyticus and E. coli.
The number of live cells of each species was determined by the
colony counting method in agar plates.
Mixed culture of P. curdlanolyticus and E. coli in an agar plate
After each cell suspension was serially diluted with sterilized
NaCl solution (0.9%, w/v) down to a cell density of 101 CFU
mL1, cell suspensions of P. curdlanolyticus and E. coli were
mixed to form suspensions with different density ratios. The cell
density of E. coli was kept at approximately 104 CFU mL1, and
This journal is ª The Royal Society of Chemistry 2009
the cell density of P. curdlanolyticus was either 104, 103, or 102
CFU mL1. Then the mixtures with different ratios were
respectively spread on TSA plates, and incubated at 30 C for
36 h. Based on the different morphology, colonies of either
P. curdlanolyticus or E. coli were distinguished visually.
cellulolytic enzymes produced by P. curdlanolyticus, whereas
lower intensity levels of fluorescence indicated either empty plugs
or plugs containing non-cellulolytic species, i.e. E. coli.
Isolation of bacterial cells in plugs by stochastic confinement and
incubation of the plugs
To culture each plug containing either P. curdlanolyticus or
E. coli, plugs were deposited31 0.5 cm apart onto a TSA plate using
a micro-aspirator (Stoelting) under a stereomicroscope (SMZ-2E,
Nikon). One spot on a TSA plate corresponded to a single plug.
An Easigrid colony template (Jencons) was attached on the
bottom of each plate to guide the deposition of plugs and to track
the locations of the deposited plugs. After deposition of plugs, the
plate was then incubated at 30 C overnight. Growth of colonies
of either P. curdlanolyticus or E. coli indicated the presence of that
species in the corresponding plug.
Plugs, approximately 10 nL in volume, were formed by using the
method described previously.20 Briefly, we formed the plugs in
a three-inlet PDMS device with 100 mm wide channels by flowing
the cell suspension diluted with TSB at approximately 5 104
CFU mL1 at 0.4 mL min1 and the fluorinated carrier fluid
(FC40 containing 0.5 mg mL1 RfOEG) at 0.5 mL min1. A long
spacer of carrier fluid was introduced via the third inlet after
every 30 plugs were formed. Plugs were collected into 200 mm I.
D. Teflon tubing, which was inserted into the device up to the
inlet junction and sealed in place with wax (Hampton Research).
After formation of plugs, the tubing was disconnected from the
PDMS device, and the ends of the tubing were sealed with wax.
Then, the tubing was incubated in a Petri dish containing 0.5 mL
of TSB media at 30 C. To maintain sterility, all tubing, devices,
and syringes used were sterilized by using 70% (v/v) ethanol, and
all solutions and media used were either autoclaved or filtered
through a PES or PTFE filter with 0.45 mm pore size.
Splitting plugs
We split each individual plug into four daughter plugs by two
steps of two-way splitting via the method described previously.18,30 As the plugs moved through the 200 mm I. D. Teflon
tubing towards the junction, each plug was split into two
daughter plugs, and each of these daughter plugs was also split
(Fig. 2c). In other words, four identical daughter plugs were split
off from each incoming plug. The daughter plugs resulting from
a particular splitting event were collected into four separate
segments of 100 mm I. D. Teflon tubing, for a total of four arrays
of daughter plugs. After splitting, the plugs in each daughter
array were recognized as copies of the initial incoming plugs, and
the cells in the initial plugs had been distributed among the
daughter plugs. The order of the daughter plugs in each array
corresponded to the original order of the incoming plugs from
which they were generated.
Cellulase assay on bacteria in an array of plugs
The synthetic substrate resorufin cellobioside (MarkerGene
fluorescent cellulase assay kit, MGT Inc.) was used to measure
the cellulolytic activity of either P. curdlanolyticus or E. coli in
culture broth. The stock solution of resorufin cellobioside (5
mM) in DMSO was diluted by the reaction buffer (100 mM
sodium acetate, pH 6) into 0.5 mM resorufin cellobioside solution. This resorufin cellobioside solution was then injected into
each plug in an array by using a PDMS T-junction with a volumetric ratio of 1 : 1. After disconnecting the tubing containing
the plugs from the PDMS device and then incubating the tubing
at room temperature for 8 h, each plug was imaged (see ESI for
details†). High intensity of red fluorescence indicated that the
fluorogenic substrate of resorufin cellobioside was cleaved by the
This journal is ª The Royal Society of Chemistry 2009
Plate culture of bacteria in an array of plugs
Cryo-preservation of bacteria in an array of plugs
For the long-term preservation of plugs, an autoclaved 40% (v/v)
glycerol stock solution was injected into each plug in an array by
using a T-junction with a volumetric ratio of 1 : 1, so that the
final concentration of glycerol in each plug was 20% (v/v). The
tubing containing plugs was frozen rapidly by being dipped in
liquid nitrogen, and then the tubing was stored in a deep freezer
at 80 C. After the frozen tubing was aseptically thawed on
a clean bench at room temperature, each plug was transferred
onto the surface of a TSA plate as described above, and then the
plate was incubated at 30 C overnight to revive cells.
Gram staining of bacteria in an array of plugs
The LIVE BacLight bacteria Gram stain kit (Molecular Probe)
was used to stain the bacteria. The staining solution was
prepared by adding 1.5 mL of 3.34 mM SYTO9 (a green fluorescent dye that stains both live, Gram-positive and live, Gramnegative bacteria) in DMSO and 1.5 mL of 4.67 mM hexidium
iodide (a red fluorescent dye that preferentially stains live,
Gram-positive bacteria) in DMSO to 1 mL of filter-sterilized
water. An array of droplets of the staining solution, each
droplet approximately 60 nL in volume, was prepared by
spotting the staining solution on a piece of cover slide immersed
in FC40 by using 300 mm I. D. Teflon tubing (Weico Wire &
Cable). Plugs, contained in 100 mm I. D. Teflon tubing, were
then deposited onto the cover slide; each plug was deposited
into one droplet of staining solution. Bacteria in each droplet
were then imaged. Green fluorescence indicated Gram-negative,
live E. coli cells, whereas red fluorescence indicated Grampositive, live P. curdlanolyticus cells.
Fluorescence In Situ hybridization (FISH) of bacteria in an array
of plugs
A 16S ribosomal-RNA targeted probe specific to P. curdlanolyticus (Pc196) was designed by Ribocon GmbH and constructed
by and purchased from biomers.net – the biopolymer factory
(Ulm, Germany) (sequence: 50 -gaa aga ttg ctc ctt ctt-30 conjugated to the fluorophore Atto550 at the 50 end). We developed
a new protocol for the detection of P. curdlanolyticus, and
optimal hybridization conditions for the probe were determined
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using P. curdlanolyticus that had been cultured in bulk in TSB
media and then fixed while the P. curdlanolyticus cells were in the
early stages of exponential growth. The solution for fixing the
cells consisted of 50% cold ethanol (4 C) and 50% RNase-free
1 PBS (145 mM NaCl, 1.4 mM NaH2PO4, 8 mM Na2HPO4, in
RNase-free water (Thermo Scientific), pH 7.4). The P. curdlanolyticus cells in fixative were then stored at 20 C for 20 h. The
fixation procedure for detection of P. curdlanolyticus in bulk was
modified as follows to detect P. curdlanolyticus isolated in plugs.
For fixation after stochastic confinement, plugs containing
bacteria grown in TSB media were merged with a stream of 100%
ethanol by using a T-junction with a volumetric ratio of 1 : 1
(Fig. 6a). The resulting plugs of 50% ethanol and 50% TSB media
were incubated at 20 C for 20 h. After fixation, plugs containing fixed bacteria were spotted on UltraStick glass slides
(Gold Seal). While most bacteria adhere to the UltraStick glass
slides, to prevent any potential cross contamination of isolated
bacterial species between spotted plugs, a PDMS membrane
containing 5% carbon and ten 5 cm wells was sealed to the
UltraStick slides, and one plug was spotted in each well (Fig. 6b).
Prior to hybridization, the hybridization buffer (900 mM NaCl,
20 mM Tris/HCl, 20% formamide, 0.1% SDS, and 5 ng mL1
Pc196 in RNase-free water) was pre-warmed at 48 C. For
hybridization, 100 mL of the buffer was added to each well
containing a plug of isolated bacteria. Bacteria were incubated in
the hybridization solution for two and a half hours at 48 C. The
hybridization solution was then removed, and bacteria were
washed for 30 minutes with washing solution (56 mM NaCl, 20
mM Tris/HCl, 5 mM EDTA, 0.01% SDS in RNase-free water) at
48 C, and next washed at room temperature with 1 PBS
buffer (pH 7.4). Following hybridization, each spot corresponding to a cataloged plug was scanned using a VT Infinity 2D array scanner confocal system with an array of 50 mm pinholes
(Visitron Systems, Germany) coupled to a Leica DMI6000
inverted microscope (Leica, Germany) and a 568 nm 2 nm
diode laser (exposure time 800 ms, gain 180). Images were
obtained with a back-thinned electron multiplier CCD camera
(16 bit, 512 512 pixels) (Hamamatsu Photonics, Japan) and
a 20 0.7 NA objective using Simple PCI software (Hamamatsu
Corportation, Japan). All stained bacteria were kept in 50%
DABCO during imaging to prevent photo-bleaching.
While the camera used did not provide truly quantitative
measurements, intensities of imaged P. curdlanolyticus and E.
coli were compared to ensure reliable identification of P. curdlanolyticus, and the intensities were analyzed using MetaMorph
Imaging System (Molecular Devices). A line scan with a scan
width of one pixel was taken across the bacteria. Data presented
is of the intensity along the line scan minus 200 a.u. of background scattering. Images were processed using Adobe Photoshop 6.0.
Results and discussion
We are interested in applying the combination of chemistrode
and stochastic confinement to study environmental microbiology
and the human microbiome. Some microbes in these environments are ‘‘unculturable’’ via traditional plate-based methods.
The media used in these traditional methods may selectively
enrich certain species in the environmental sample, allowing
2156 | Lab Chip, 2009, 9, 2153–2162
them to out-compete other species. The losing species in this
competitive interaction are not recovered from plates and
therefore deemed ‘‘unculturable’’. However, it may be the case
that the growth of these ‘‘unculturable’’ species strongly relies on
their original media,11 which may contain specific amounts of
certain materials or molecules which are required for growth. If
so, culturing these ‘‘unculturable’’ microbes should require using
the original sample from the environment, and then incubating
the cells in the original media, rather than on plates. One of the
challenges of using the original sample from the environment is
that samples from some environments – such as soil, sediments,
blood, and the human gut – may adhere to and foul or occlude
the sampling devices. Plugs can be used to transport solids reliably, including suspensions of inorganic nanoparticles,32 protein
microcrystals,33 agglutinated red blood cells and clotted blood.34
To sample microbes directly from their environment, in their
original media, and without fouling of the device, we used
a modified chemistrode to form plugs immediately as the sample
flows into the tip (Fig. 1a). This modified chemistrode was
demonstrated by sampling from soil slurry (Fig. 1b). A similar
device has been recently described,35 and we thank the reviewer
for pointing out this reference to us.
Having demonstrated that the modified chemistrode can
successfully sample directly from the environment, we then
tested the rest of the approach (Fig. 2) by forming plugs using
a device similar to the one that was previously used for
stochastic confinement.20 With a mixture of two fluorescencelabeled strains of E. coli: GFP-labeled E. coli and RFP-labeled
E. coli in equal proportions, we used the principle of stochastic
confinement to isolate single bacterial cells into individual
plugs. When the number of plugs generated was much higher
than the number of cells present initially, among those plugs
containing cells, most contained only cells of GFP-labeled
E. coli or only cells of RFP-labeled E. coli. The statistical
property of this process can be described by the Poisson
distribution.27 Previous work has shown that encapsulation of
cells in plugs does not introduce artifacts to cell growth,23 and
we confirmed that single cells, isolated in plugs and incubated,
formed populations with growth rates similar to those observed
for cells in bulk solution (Fig. S2†).
After we incubated the plugs to allow time for a few divisions
of the original cell within a plug (Fig. 2b), we split each of the
plugs in the original array to create four identical, parallel
daughter arrays (Fig. 2c). There was no cross-contamination
between plugs during the splitting, because the plugs were well
separated from each other, as well as from the surfaces of the
tubing and the PDMS devices, by the carrier fluid. We calculated
the statistical probability of the resultant distribution of cells in
the four daughter plugs, assuming that all cells in each initial plug
had an equal probability of being distributed into each of the
daughter plugs. After five cycles of divisions from a single cell
(i.e. when there are 32 cells in a plug), there is greater than 99%
probability of having at least one cell in each daughter plug. We
were also concerned that cells of some bacterial species would
form clusters and not split evenly among the daughter plugs. We
experimentally confirmed that gentle shear in the plugs (average
shear rate 60 s1) was sufficient to break up any clusters of all
strains of either Escherichia coli (E. coli) or Paenibacillus curdlanolyticus used for this paper. Thus, we presume that splitting
This journal is ª The Royal Society of Chemistry 2009
Fig. 1 A modified chemistrode performs sampling of a substrate and enables formation of plugs at the sampling site. (a) A schematic drawing of the
design of the chemistrode, which consists of a piece of hydrophilic silica capillary with a piece of hydrophobic Teflon tubing inserted inside. The carrier
fluid was delivered to the tip of the probe through the space between the silica capillary and the Teflon tubing. Aspiration at flow rate higher than the flow
rate at which the carrier is delivered removed both the carrier fluid and the aqueous sample into the Teflon tubing, and the aqueous stream was
segmented into plugs by the carrier fluid at the entrance of the Teflon tubing. The carrier fluid was contained inside the chemistrode by capillary forces
that keep the carrier fluid adherent to the Teflon walls. A photograph on the lower left shows the tip of the chemistrode sampling from water.
A photograph on the lower right shows plugs of a solution of red dye transported by the chemistrode. (b) A schematic (left) of the chemistrode sampling
from soil slurry; and the sample plugs are shown in the tubing downstream. A series of time-lapse brightfield images (right) show a sample plug
containing soil particles passing through the tubing. The red dot is a stationary reference point.
should reliably generate parallel copies of the bacterial populations for multiple tests.
Having demonstrated stochastic isolation and incubation of
individual cells from an equal-ratio, multi-species mixture, we
then tested whether this approach can be used to do the same for
cells of a rare species in a multi-species mixture. In this context,
P. curdlanolyticus was chosen as the rare, relatively slow-growing
species, and wild-type E. coli was chosen as the abundant,
rapidly-growing species. P. curdlanolyticus cells produce and
secrete cellulolytic enzymes, a class of enzymes interesting for
conversion of biomass into biofuels; whereas, the E. coli species
that we used does not produce any similar enzymes to cleave
cellulose.
We prepared mixtures of E. coli and P. curdlanolyticus at
different ratios of viable cell numbers – 1.5 : 1, 2 : 1, 15 : 1, 20 : 1,
and 200 : 1 – and spread suspensions of these mixtures on TSA
plates. We were able to detect P. curdlanolyticus colonies visually, based on differences in morphology of colonies, in plates
spread with suspensions of the mixture with the ratio of either
1.5 : 1 (Fig. 3a) or 2 : 1 (Fig. 3c). However, we could not detect
any P. curdlanolyticus colonies in plates with the ratio of 15 : 1
This journal is ª The Royal Society of Chemistry 2009
(Fig. 3b), 20 : 1 and 200 : 1 (Fig. 3c), indicating that E. coli was
out-competing P. curdlanolyticus on the TSA plates. This result
confirms the known difficulty of isolating rare species present at
low abundance using conventional plating methods. Here, when
the ratio of E. coli to P. curdlanolyticus was above 15 : 1, no cells
of the rare species were recovered.
Then, we isolated the cells in the mixture into individual plugs
and incubated the cells of E. coli and P. curdlanolyticus in plugs.
We were able to recover P. curdlanolyticus cells that successfully
grew up to hundreds of cells in all cases; we tested ratios from
1 : 1 to 40 : 1 (Fig. 3d and 3e). This observation is consistent with
what we expected, because the individual species were separated
into different plugs so that the slow-growing species (i.e.
P. curdlanolyticus strain) can have an opportunity to grow
without any competition with the fast-growing species (i.e. E. coli
strain). The fraction of the recovered P. curdlanolyticus was
always lower than 100%, because there is a minor probability
that both a P. curdlanolyticus cell and an E. coli cell are isolated
together in some plugs, according to the Poisson distribution. In
such a case, the E. coli cells would dominate the growth in those
plugs, and the P. curdlanolyticus cells in those plugs could not be
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Fig. 2 The chemistrode combined with stochastic confinement can be
used to (a) isolate individual cells in a multi-species mixture by stochastically encapsulating the cells in an array of individual plugs, (b) incubate
the plugs to grow colonies of the isolated cells without cross-species
interference, (c) split the plugs into arrays of identical daughter plugs to
perform multiple tests in parallel, such as identification and functional
tests. To demonstrate, individual bacterial cells of either GFP-labeled
E. coli or RFP-labeled E. coli were stochastically isolated from a mixture
of both E. coli strains into plugs. Incubation of the plugs enabled these
isolated single bacterial cells to grow. The three images in (b) show three
adjacent plugs in an array. The first plug contains GFP-labeled E. coli,
the second plug is empty, and the third plug contains RFP-labeled E. coli.
These plugs were incubated for 6 h after formation, and the bacterial cell
in each individual plug (if present) divided and grew. After incubation,
each plug was split into four identical daughter arrays (c). The four leftmost images in (c) show the daughter plugs generated by splitting the leftmost plug in (b). The four central images in (c) show the daughter plugs
generated by splitting the central plug in (b). The four right-most images
in (c) show the daughter plugs generated by splitting the right-most plug
in (b).
recovered. The fraction of P. curdlanolyticus cells that were
recovered was variable among different experiments, presumably
because of the stochasticity of the isolation of cells into plugs.
The culturing method of ‘‘dilution to extinction’’ also uses the
principle of stochastic isolation.14,15 However, in these methods
there is a very low starting concentration of the inoculum (100
CFU mL1). On the other hand, encapsulating a single bacterium
in a plug with a volume of 10 nL provides a cell density of 105
mL1 in a single plug. Increasing the inoculum density (e.g. 105
2158 | Lab Chip, 2009, 9, 2153–2162
CFU mL1) allows a dramatic reduction in the culturing time
required to reach detectable cell densities. Creating high inoculum density by isolating individual bacterial cells in plugs is
especially important for those bacterial species that have slower
division rate at lower concentration,36 because the rapid accumulation of the molecules or enzymes in the confined environment of the plug may enhance growth of cells.29 Furthermore,
plugs require much lower volumes of reagents for the isolation
and the cultivation of bacterial samples. Thus, plug-based
methods that rely on stochastic isolation have many advantages
over the culturing method of ‘‘dilution to extinction’’.
Methods with gel microdroplets (GMDs) also use stochastic
confinement to isolate single cells,10,17 and isolation by GMDs
may be useful for separating and culturing symbiotic species, as
compounds can diffuse between GMDs.10 Some other microfabrication-based methods also have been developed to manipulate and analyze isolated single cells,37–39 however, using plugs
allows splitting of the plugs to form identical copies for analyses
with multiple techniques in parallel.
Having demonstrated stochastic isolation and incubation of
rare cells in a mixture, we next tested whether these methods
could be combined with splitting to create parallel arrays for
further testing (Fig. 4). We started with a mixture of E. coli and
P. curdlanolyticus at a concentration ratio of 40 : 1, which does
not result in recovery of P. curdlanolyticus colonies when the
mixture is spread on plates. After isolation and incubation in
approximately 1000 plugs, we split 100 plugs into four daughter
arrays. Each daughter array also contained 100 plugs, all of
which were copies of the original plugs. We then used each array
of plugs to perform one of four different tests.
The first daughter array of plugs was used for the cellulase
assay. Fluorescence in each plug was detected after each plug
was injected with a solution of the fluorogenic substrate resorufin cellobiosides and incubated at room temperature for 8 h.
Plugs containing P. curdlanolyticus showed more than two times
higher fluorescence than plugs containing E. coli, and more than
four times higher fluorescence than empty plugs (Fig. 4a,
Fig. S3†). Injection of reagents into plugs is well established.18,32,40 Plugs in the second daughter array were deposited
onto a fresh TSA plate, and then incubated at 30 C for 20 h to
grow individual colonies. We confirmed that pure colonies of
either P. curdlanolyticus or E. coli grew on the surface of the
TSA plate where we deposited the corresponding plugs containing either P. curdlanolyticus or E. coli (Fig. 4b). Plugs in the
third daughter array were used for cryo-preservation. After
injection of glycerol, the plugs were frozen by using liquid
nitrogen, and then they were stored at 80 C for one day. To
test the recovery of the viability of the cells in the cryo-preserved
plugs, those plugs were thawed on the second day and deposited
onto a fresh TSA plate for incubation. Colonies grew on all the
locations on the surface of the TSA plate where plugs containing cells were deposited (Fig. 4c). The fourth daughter array
of plugs was used for Gram staining. We confirmed that the
Gram-positive P. curdlanolyticus species was stained in red, and
the Gram-negative E. coli species was stained in green (Fig. 4d)
by the fluorescent dye used here.
In this experiment, we developed a new method for analyzing
plugs: an array of droplets is deposited onto a surface, and
individual plugs are injected into the droplets of the surface array
This journal is ª The Royal Society of Chemistry 2009
Fig. 3 Rare individual cells in a mixture were isolated by stochastic confinement at ratios much lower than those that were achievable using plate-based
methods. (a) Colonies of both E. coli and P. curdlanolyticus were observed after spreading a mixture of E. coli and P. curdlanolyticus cells, with the ratio
of cell density at 1.5 : 1, onto a TSA plate and incubating the plate for 36 h. (b) Colonies of only E. coli cells were observed after spreading a mixture of E.
coli and P. curdlanolyticus cells, with the ratio of cell density at 15 : 1, onto a TSA plate and incubating the plate 36 h. No P. curdlanolyticus colonies were
observed after 36 h. (c) A plot of the fraction of colonies recovered after spreading mixtures of E. coli and P. curdlanolyticus with different cell density
ratios on TSA plates and incubating the plates for 36 h. The fraction recovered is defined as the ratio of the number of colonies observed on the plates to
the number of colonies expected, based on the number of viable cells of each organism, measured by culturing pure cell suspensions prior to mixing them.
Error bars denote standard deviations (n ¼ 2). (d) Bright field images show the population of P. curdlanolyticus cells grown from the cell(s) isolated by
stochastic confinement in a plug. (e) A plot of the fraction of cells recovered after mixtures of E. coli and P. curdlanolyticus with different concentration
ratios were isolated in plugs and incubated. The fraction of cells recovered is defined as the ratio of the number of plugs with each species to the number
of plugs expected, based on the number of viable cells of each organism, measured by culturing pure cell suspensions prior to mixing them. See detailed
data in Table S1†. Pc indicates P. curdlanolyticus.
Fig. 4 Populations grown from the isolated single cells were split for parallel tests. Individual cells in a mixture of E. coli and P. curdlanolyticus (ratio of
cell density, 40 : 1) were isolated stochastically in plugs and incubated. Then the plugs were split into four daughter arrays which were used for (a)
cellulase assay, (b) cultivation, (c) cryo-preservation, and (d) Gram staining, respectively (see the text/ESI†). Each row of microscopic images on the right
shows the results of one of the four tests from three sequential plugs in each array. Each column, marked with colored stars, shows the test results for
daughters, i.e. clones, of a bacterium from a single plug: the green stars trace the four daughter plugs split from a plug containing E. coli, the red stars
trace the daughter plugs from a plug containing P. curdlanolyticus, and the blue stars trace the daughter plugs from an empty plug. The abbreviation LN
in (c) means liquid nitrogen. Pc indicates P. curdlanolyticus.
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Lab Chip, 2009, 9, 2153–2162 | 2159
to induce a reaction. We used this method to perform Gram
staining of cells in plugs (Fig. 5). After immersing a piece of glass
slide in fluorocarbon, an array of aqueous droplets of the reagent
for analysis (here it was Gram staining solution) was first
generated on the slide surface (Fig. 5a). Next, each plug in the
tubing was deposited into one droplet of reagent, and due to
surface tension, the plugs and the droplets merged reliably
(Fig. 5b, inset). Readout of reactions in the droplets was imaged
optically from the bottom of the slide. Because there was no
curved surface in the light pathway (Fig. 5c), aberrations
common in imaging of plugs in tubing were eliminated. Two
common problems of injecting reagents into plugs by using
a PDMS T-junction are that volumetric injection ratio is
limited,40 and that many compounds in the reagents tend to be
adsorbed on the PDMS surface. This method circumvents these
problems while still enabling analysis with throughput up to
103 plugs per trial; even higher throughput can potentially be
achieved by integration with automatic control. Moreover,
immersion in excess fluorocarbon slows down the evaporation of
the nanoliter droplets and enables long reaction times – potentially up to a week if at room temperature. This method has the
potential to be generally adopted for analyzing the contents of
plugs.
An important feature of this approach of combining the
chemistrode with stochastic confinement is that splitting of the
plugs not only enables performing multiple parallel tests
immediately after incubation of the cells in the plugs, but also
allows tracing each plug in a daughter array back to the initial
plug from which it was split. Because the order of the original
plugs is preserved in the order of the daughter plugs, plugs in
parallel daughter arrays can be traced by encoding patterns in
the sequence of plugs when they are formed. In the experiments
for this paper, a long carrier fluid spacing was introduced
between every 30 plugs for the identification of sequences of
plugs. Thus, the four arrays were reliably aligned, and any
potential errors in the alignment, such as might occur if a plug
were to split or two plugs were to coalesce, did not propagate.
For tests that required transferring plugs from the tubing onto
different plates, the locations of the deposited plugs were
tracked by using grids. By tracing the sequences of plugs, we
matched the order of the four different plugs in the four
different arrays that were used for the four different tests. On
one hand, rapid classification or identification tests can locate
particular plugs containing cells of interest in the array, so that
these plugs can be collected for culture. On the other hand, the
ability to preserve viable cells in plugs allows for culture of cells
long after the splitting event and after knowing the results of
a parallel identification test on another daughter array. The
splitting also enables the use of incompatible techniques on
identical copies of cell populations from the same original single
cell. For example, identification techniques such as gene
sequencing and FISH require killing of the cells, while live cells
are required for cultivation and functional tests as the cellulase
assay.
Finally, we demonstrated that FISH can be used to identify
P. curdlanolyticus cells in one of the identical daughter arrays
while using the cells in another daughter array for cultivation
(Fig. 6). We developed a new protocol to detect P. curdlanolyticus in bulk and modified this protocol (see Experimental) to
detect P. curdlanolyticus isolated in plugs. After isolating and
incubating a mixture of P. curdlanolyticus and E. coli with
a concentration ratio of 1 : 1 in approximately 200 plugs, all
plugs were split into four identical daughter arrays. Cells in
plugs from the first daughter array were fixed by injecting 100%
ethanol into each plug using a T-junction, resulting in 50%
ethanol and 50% TSB media (v/v) in each of the plugs. Then the
plugs were stored at 20 C for 20 h and fixation took place
(Fig. 6a). After fixation, ten plugs were spotted into wells of
a PDMS membrane sealed to an UltraStick glass slide (Fig. 6b).
Following staining with a 16S ribosomal-RNA probe specific to
P. curdlanolyticus (Pc196) (see Experimental), plug 6 (Fig. 6c)
was identified as containing P. curdlanolyticus. Plug 4 was
identified as containing a different species (E. coli), judging by
weak non-specific fluorescence (Fig. 6c). Plug 3 is an example of
a blank plug (Fig. 6c). Using this protocol, the fluorescence
intensity of P. curdlanolyticus stained with the Pc196 probe
was 9000 a.u. above background, and fluorescence of E. coli
stained with the same probe was 100 a.u. above background.
This 90 : 1 ratio of intensities facilitated the identification of
P. curdlanolyticus (Fig. 6d). Fluorescence intensity was similar
to that of controls where the procedure was performed in bulk
(i.e. outside of plugs), indicating that FISH staining from plugs
can be performed without loss of staining quality (Fig. S4†).
Plugs in the second daughter array were spotted and cultured on
a culture plate. Colonies of P. curdlanolyticus also grew at the
location of plug 6 (Fig. 6e).
Fig. 5 A schematic drawing of Gram staining, which was performed on a glass slide immersed in fluorocarbon. (a) A two-dimensional array of droplets
of the staining solution for the Gram test, a mixture of SYTO9 and hexidium iodide, was first spotted onto a piece of glass slide immersed in fluorocarbon (FC40). (b) The plugs for the test were then deposited into the droplets of the staining solution. One plug was deposited into each droplet of the
staining solution. (c) The fluorescence of the stained cells in each droplet was observed from the bottom of the slide by using an epi-fluorescence
microscope. The slide was immersed in FC40 during the whole process so that there was no significant evaporation of aqueous droplets.
2160 | Lab Chip, 2009, 9, 2153–2162
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Fig. 6 Parallel live cultivation and killing identification by Fluorescence In Situ Hybridization (FISH) on clones of the same cell. FISH was used to
identify P. curdlanolyticus cells in one of the identical daughter arrays while using the cells in another daughter array for cultivation. (a) A schematic
drawing of fixation of the cells in an array of plugs for FISH by injection of ethanol into each plug using a T-junction. (b) A schematic drawing of
attaching a PDMS membrane containing 5% carbon with holes onto an UltraStick glass slide to form staining wells for FISH, into which the plugs
containing fixed cells were then deposited. (c) Ten plugs in a daughter array were deposited and stained by FISH using the probe Pc196 (see Experimental). P. curdlanolyticus cells contained in plug 6 (shown) and plug 5 (not shown) were identified. Plug 3 and plug 4 are examples of blank and E. coli
containing plugs, respectively. A parallel array of daughter plugs was used for cultivation. (d) Fluorescence intensity (a.u.) of the stained cells in plugs
shown in (c), as measured by an array scanner confocal microscopy. (e) A colony of P. curdlanolyticus that grew after culturing plug 6 from the second
array. Pc indicates P. curdlanolyticus.
Conclusions
In this paper, we demonstrate how plug-based microfluidic
approaches can be used to accomplish a set of single-cell
microbiologic processing: from sampling directly from an
environment, to isolating single cells from a multi-species
mixture, to identifying cells of interest by killing methods
while at the same time providing their clonal copies for
functional testing, culturing, or preservation. Sampling
directly from the environment with high spatial resolution, as
provided by the chemistrode, is attractive for understanding
spatial structures present in natural and synthetic microbial
communities.41,42
This approach requires that isolated cells are culturable individually in plugs. As a result, it is suitable for working with any
cells that do not require symbiotic microorganisms for growth,
and it is especially useful for attaining rare species in mixtures
that are dominated by competitive interactions, or for cells that
show enhanced growth in confined volumes.29
The modified chemistrode presented in this paper would
facilitate culturing isolated cells in their original media, which
may contain compounds required for growth. Even if the initial
growth in plugs is minimal (just a few divisions), species of
interest can be identified by FISH or by other genetic methods,
such as 16S rRNA sequencing, which has been applied to identify
single cells isolated on a microchip.39 After identification, alternative techniques and conditions can be used to successfully
culture the sample of the species preserved in the parallel array.
This journal is ª The Royal Society of Chemistry 2009
For example, the hybrid method43 could be used to generate large
numbers of different conditions in plugs to screen optimal
growth condition.
The microfluidic approach described in this paper would be
potentially useful for studies in environmental microbiology
and of the human microbiome or for diagnostics. We demonstrated the approach here for microbes, but it may be applicable
for other cells in addition to bacteria. For example, it may be
used to propagate and analyze immune cells after isolation from
patients. Similarly, we demonstrated the integration of FISH
for identification of rare bacterial species with stochastic
confinement using P. curdlanolyticus as the rare species, but
this approach may be applicable for detection of other
microbes or cells in single or low numbers, including Grampositive bacteria, Gram-negative bacteria, or mammalian cells
as is performed in prenatal and cancer diagnostics. This
approach would also complement current methods for FISH
detection.8,9,44–47
Acknowledgements
This work was supported by the National Institutes of Health
Director’s Pioneer Award 1DP1OD003584. We thank Mitchell
L. Sogin for helpful discussions, Jessica Mark Welch and Gary
Borisy for advice on FISH, Delai Chen for advice on the
chemistrode, and Elizabeth B. Haney for contributions to writing
and editing this manuscript.
Lab Chip, 2009, 9, 2153–2162 | 2161
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